Mammalian genomes encode thousands of long non-coding (lnc) RNAs, many with important functions including the regulation of gene expression, yet, how lncRNAs function remains largely unexplored. The lncRNA Xist provides a remarkable model to investigate the function of lncRNAs in gene regulation, as it spreads from its site of transcription on the X chromosome over the entire chromosome in cis to induce gene silencing, alter chromatin state, and modulate the three-dimensional chromosome architecture in the process of X-chromosome inactivation (XCI). XCI is fundamentally important for female mammalian development but, despite its critical role, the mechanisms by which Xist carries out the various tasks associated with XCI still remain largely unclear. Recently, our and other labs proposed that Xist fulfills its different roles during XCI, such as gene silencing, chromatin association, spreading, recruitment of repressive chromatin regulators, membrane-less compartment formation, through different RNA domains, which in turn recruit different proteins. The 17kb long Xist RNA consists of a series of conserved repeats, termed A-F, as well as intervening non-repeat regions. Both repeat and non-repeat regions have been demonstrated to bind proteins, so both types of sequences can form functional domains. However, except for the A-repeat, which is now known to mediate silencing by recruitment of the proteins SPEN and RBM15, the function of nearly all other Xist sequences is still unknown. In this proposal, I will determine the Xist domains required for the initiation and maintenance of XCI in female mESCs. I will characterize an embryonic stem cell (ESC) line that carries a deletion of the C-repeat of Xist, which I have generated, to determine the role that this domain plays in XCI. In addition, I will utilize a unique XCI reporter system that I have developed in mouse ESCs, to systematically identify additional functional Xist domains and begin to understand their function. Furthermore, I propose to use a forced recruitment system and a novel protein degradation system to characterize how Xist interactors contribute to XCI. Through these experiments, I will be able to examine the molecular mechanisms of specific Xist-protein interactions, to reveal the means by which Xist, through its RNA domains, integrates different functions. The proposed studies will provide new paradigms for regulation of gene expression by lncRNAs, reveal important insights into the molecular regulation of XCI by Xist, and uncover novel roles of RNA-binding proteins.